Single-stranded circular DNA is useful in many areas of biotechnology, both of an experimental as well as commercial nature. One important such use is as a substrate for rolling circle DNA replication, in which a single-stranded circle of DNA is used as a template by a DNA polymerase to produce a long, unbroken single strand of repeating DNA sequence. Single-stranded circular DNAs also find use in single molecule real-time DNA sequencing technologies capable of repeatedly re-sequencing a single piece of double stranded DNA, using a single-stranded circular DNA molecule as template. Single-stranded circular DNAs also find use in microfluidic devices designed to prepare sequence libraries for next-generation sequencing technologies.
Methods for the creation of single-stranded circular DNA molecules have been described (U.S. Pat. No. 7,041,480; WO/2009/120372; National Human Genome Research Institute's “Advanced Sequencing Technology Development Meeting” Stephen Turner, Apr. 1, 2009; “Rapid Library Preparation and Single-molecule (SMRT) Sequencing Enable Same Day Influenza A Genome Sequence Analysis,” Poster, AGBT conference, Feb. 24-27, 2010; herein incorporated by reference in their entireties). These methods include ligation of hairpin adapters to the terminal ends of double stranded DNA fragments one wishes to sequence, and ligation of hairpin monomers containing the desired DNA sequence. However, these methods are complex, requiring multiple steps and significant amounts of time (e.g. multiple hours). What is needed are simple, rapid, and efficient PCR methods to produce single-stranded circular DNA from a target sequence of interest.